1. Field of the Invention
This invention relates to novel mutants of the first Kunitz domain (K.sub.1) of the human lipoprotein-associated coagulation inhibitor LACI, which inhibit plasmin. The invention also relates to other modified Kunitz domains that inhibit plasmin and to other plasmin inhibitors.
2. Description of the Background Art
The agent mainly responsible for fibrinolysis is plasmin, the activated form of plasminogen. Many substances can activate plasminogen, including activated Hageman factor, streptokinase, urokinase (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (pKA). pKA is both an activator of the zymogen form of urokinase and a direct plasminogen activator.
Plasmin is undetectable in normal circulating blood, but plasminogen, the zymogen, is present at about 3 .mu.M. An additional, unmeasured amount of plasminogen is bound to fibrin and other components of the extracellular matrix and cell surfaces. Normal blood contains the physiological inhibitor of plasmin, .alpha..sub.2 -plasmin inhibitor (.alpha..sub.2 -PI), at about 2 .mu.M. Plasmin and .alpha..sub.2 -PI form a 1:1 complex. Matrix or cell bound-plasmin is relatively inaccessible to inhibition by .alpha..sub.2 -PI. Thus, activation of plasmin can exceed the neutralizing capacity of .alpha..sub.2 -PI causing a profibrinolytic state.
Plasmin, once formed:
i. degrades fibrin clots, sometimes prematurely; PA1 ii. digests fibrinogen (the building material of clots) impairing hemostasis by causing formation of friable, easily lysed clots from the degradation products, and inhibition of platelet adhesion/aggregation by the fibrinogen degradation products; PA1 iii. interacts directly with platelets to cleave glycoproteins Ib and IIb/IIIa preventing adhesion to injured endothelium in areas of high shear blood flow and impairing the aggregation response needed for platelet plug formation (ADEL86); PA1 iv. proteolytically inactivates enzymes in the extrinsic coagulation pathway further promoting a prolytic state. Robbins (ROBB87) reviewed the plasminogen-plasmin system in detail. ROBB87 and references cited therein are hereby incorporated by reference. PA1 i. Neutralization of relevant target fibrinolytic enzyme(s); PA1 ii. High affinity binding to target enzymes to minimize dose; PA1 iii. High specificity for target, to reduce side effects; and PA1 iv. High degree of similarity to human protein to minimize potential immunogenicity and organ/tissue toxicity. PA1 1) the K.sub.i for plasmin is at most 20 nM, preferably not more than about 5 nM, more preferably not more than about 300 pM, and most preferably, not more than about 100 pM, PA1 2) the inhibitor comprises a Kunitz domain meeting the requirements shown in Table 14 with residues number by reference to BPTI, PA1 3) at the Kunitz domain positions 12-21 and 32-39 one of the amino-acid types listed for that position in Table 15, and PA1 4) the inhibitor is more similar in amino-acid sequence to a reference sequence selected from the group SPI11, SPI15, SPI08, SPI23, SPI51, SPI47, QS4, NS4, Human LACI-K2, Human LACI-K3, Human collagen .alpha.3 KuDom, Human TFPI-2 DOMAIN 1, Human TFPI-2 DOMAIN 2, Human TFPI-2 DOMAIN 3, HUMAN ITI-K1, Human ITI-K2, HUMAN PROTEASE NEXIN-II, Human APP-I, DPI-1.1.1, DPI-1. 1.2, DPI-1.1.3, DPI-1.2.1, DPI-1.3.1, DPI-2.1, DPI-3.1.1, DPI-3.2.1, DPI-3.3.1, DPI-4.1.1, DPI-4.2.1, DPI-4.2.2, DPI-4.2.3, DPI-4.2.4, DPI-4.2.5, DPI-5.1, DPI-5.2, DPI-6.1, DPI-6.2 than is the amino acid sequence of said Kunitz domain to the sequence of BPTI. PA1 (a) conservative substitutions of amino acids as defined in Table 9; and PA1 (b) single or multiple insertions or deletions of amino acids at termini, at domain boundaries, in loops, or in other segments of relatively high mobility. PA1 a) nonbiological synthesis by sequential coupling of components, e.g. amino acids, PA1 b) production by recombinant DNA techniques in suitable host cells, and PA1 c) semisynthesis, for example, by removal of undesired sequences from LACI-K1 and coupling of synthetic replacement sequences. PA1 i) Inadequate affinity and/or specificity; PA1 ii) Poor penetration to target sites; PA1 iii) Slow clearance from nontarget sites; PA1 iv) Immunogenicity (most are murine); and PA1 v) High production cost and poor stability.